Week of 10/28/13 - 11/1/13
More pNIC-Bsa4 was cut and ran on a gel to ensure the plasmid was indeed cut. Afterwards, another attempt at cloning was performed using the PCR squared reagents previously purified. The plates were left to incubate overnight and will be checked for bacterial growth tomorrow.

Figure 18: Gel of cut pNIC-Bsa4 samples

Week of 10/21/13 - 10/25/13
To attempt cloning once more, more secondary PCR and PCR squared was needed. Two tubes of secondary PCR was created and ran on a 1% agarose gel with a 100bp ladder. Four PCR squared samples were made using both of the secondary PCR's. The gel after PCR squared showed contamination in the 2nd secondary PCR sample, so only the 1st secondary PCR was used for clean up and cloning.

Figure 16: Two secondary PCR samples created for cloning attempt 4

Figure 17: Gel of PCR squared of the two secondary PCR samples previously made. Lanes 2-5 were from the first 2nd PCR and lanes 6-9 were samples from the second 2nd PCR samples

Week of 10/14/13 - 10/18/13
Nice work on the captions and analysis. Did you submit anything to sequencing? Also where are you with virtual work? Thank you. -Max 10/21/13
Transformation was attempted again using the recently cut pNIC-Bsa4 and gel extraction product since no colonies grew on the last plate. This time, colonies did grow after two days of incubation in the 37 degree incubator. The colonies were large, slightly yellow-orange in color, and seemed to cluster together which unfortunately suggested that the plates were contaminated. Thus, the plates were discarded and another attempt at cloning is necessary. In order to proceed, new pNIC-Bsa4 was cut with restrictive enzymes and underwent PCR clean up to yield higher concentrations. Hopefully with a higher pNIC-Bsa4 concentration, a positive clone will result.

Figure 15: Plates with unidentified growth after two days of incubation in the 37 degree Celsius incubator

Figure 16: Nanodrop of new pNIC-Bsa4 tube 1 after being cut with restrictive enzymes and PCR clean up

Figure 17: Nanodrop image of new pNIC-Bsa4 tube 2 after being cut with restrictive enzymes and PCR clean up

Week of 10/7/13 - 10/11/13
Since no colonies grew last time, another attempt at cloning was performed. There were no more reagents to use, so more pNIC-Bsa4 and PCR product needed to be made. The concentrations of the pNIC-Bsa4 cut with restrictive enzymes after PCR clean up were very low. 100 ng of each tube were ran on a gel to ensure that the pNIC-Bsa4 was throughly cut. The gel resulted in positive results because it had two bands in the correct lengths. PCR clean up was performed on eight tubes of PCR squared products. The product of the PCR clean up was then placed in another gel to be used for gel extraction. Performing both PCR clean up and gel extraction caused the concentration of the PCR product to be very low. I proceeded to the cloning process nevertheless.

Figure 11: Nanodrop image of pNIC-Bsa4 tube 1 concentration after cutting with restriction enzymes

Figure 12: Nanodrop image of pNIC-Bsa4 tube 2 with a low concentration after cutting with restrictive enzymes

Figure 13: Agarose gel with 100 ng of cut pNIC-Bsa4 tube 1 in lane 2 and cut pNIC-Bsa4 tube 2 in lane 3 with a 1kb DNA ladder in lane 1

Figure 14: Gel image of PCR squared clean up products before proceeding to gel extraction

Week of 9/30/13 - 10/5/13**

• Good analysis but try to add captions also. Those are important to getting quick information across. Thank you. -Max 10/07/20131

Using the pNIC-Bsa4 cut from last week, cloning transformation was done. But after 2 days of incubation, no colonies grew. All of the times in the transformation protocol were doubled. That could have been a reason why the colonies did not grow; the cells were heat shocked for too long.Tube A plate had 2 uL of accepting vector and 4 uL of insert while Tube B plate had 1 uL of accepting vector and 9 uL of insert.

Figure 10: Results of cloning transformation after second day of incubation in 37 degree celcius. Tube A plate had 2uL of accepting vector and 4uL of insert while Tube B plate had 1 uL of accepting vector and 9uL of insert

Week of 9/23/13 - 9/27/13
The results of gel extraction was nanopured to yield a high concentration of 451.4 ng/ul. PNIC-Bsa4 was cut with restrictive enzymes for the cloning procedures.

Figure 8: Four PCR squared tubes were collected and gel extraction was performed in order to yield a high concentration

Figure 9: Nanodrop image of pNIC-Bsa4 cut with restrictive enzymes

Week 3 & 4 9-Sep - 22-Sep
Katherine - ok keep moving your cloning foward - we want yours to be completed so that you can move forward. - Dr. B 100113
Week of 9/16/13 - 9/20/13
PCR squared was made using the secondary PCR from the previous week. Then PCR squared clean up was performed on 4 samples, but yielded very low nanodrop results due to a mistake that occured during the clean up process. Nanopure was added instead of the binding solution, so the DNA was not collected in the filter. The PCR squared protocol was doubled to create 8 samples. The samples were ran again and then used for gel extraction in order to gather a larger amount of DNA. The next steps would include using the gel extraction for cloning.

Figure 5: Nanodrop trial 1 of PCR squared after PCR cleanup

Figure 6: Nanodrop trial 2 of PCR squared after PCR cleanup

Since the concentration of the PCR squared clean up was too small, gel extraction was performed. There was no ladder to compare the sample with, but all of the lanes seem to have DNA with the same amount of base pairs. The gel was cut and underwent extraction.

Figure 7: Agarose gel with six lanes of PCR squared product used for gel extraction

Week of 9/9/13 - 9/13/13
Primary PCR and Secondary PCR was redone using the oligo mix made in the summer. All the samples were ran on a gel. The primary PCR did not show up on the gel, but the secondary PCR did with the right amount of base pairs. This could have been due to the primary PCR not having enough DNA to show up on the gel, but it had enough to be amplified. Also, Midi-prep was performed on pNIC-Bsa4 pellets. Then it was nanodropped and sent to DNA sequencing for future cloning. The pNIC-Bsa4 resulted in 90% coverage and 98% identity.
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Figure 1: First nanodrop result of pNIC-Bsa4 after midi-prepped was performed

Figure 2: Nanodrop results of pNIC-Bsa4 after midi-prep was performed trial 2

The average of the two trials is 83.9 ng/ul. Two samples, one with pLIC-forward primer and one with pLIC-reverse primer, was sent to the DNA sequencing lab.
The blast results are displayed below.

Figure 3: Blast result of midi-prepped pNIC-Bsa4 with 90% coverage and 98% identity

Figure 4: Gel of primary PCR (lane 2) and secondary PCR (lane 3) with 100 bp ladder in lane 1

The primary PCR did not show up most likely due to such a low amount of DNA. There is a slightly faint smear near the bottom of lane 2. This may be due to a degrading oligo mix since the mix made during the summer was used. Since it does not take much DNA to be amplified through PCR, the secondary PCR showed results with the right amount of base pairs ~ 900.
Week 1 & 2**
The DNA sequencing results from the pNIC-Bsa4 cloning were analyzed and blasted. The highest cloning result was 8% coverage and 100% identity. Since the results were very low and had no signs of the inserted vector, a new oligo mix was created using pfSTPP primers. A homology model was started using the SWISS model workspace and a PyMol refresher was completed as well.

Fall 2103

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Week Two: - Katherine -

DNA sequencing result and PCR result? Also - what are your Nanodrops off? (you can put the name of the sample in the Sample ID box so that when you print it - it will already be there). - Dr. B

Agarose gel preparation 6/12/13

Week One:

Transformation Efficiency 6/5/13

Figure 1. Transformation of E. coli with 25ng pNIC-BSA4 (NEB 5 alpha) grown on LB + Kan agar plates

Figure 1. Transformation of E. coli with 5ng pNIC-BSA4 (NEB 5 alpha) grown on LB + Kan agar plates

Figure 1. Transformation of E. coli with 1 ng pNIC-BSA4 (NEB 5 alpha) grown on LB + Kan agar plates

Nanodrop 6/04/13